Drosophila Trithorax-related (Trr), human MLL3 and MLL4 are homologs of a family of SET domain-containing proteins that constitute enzymes with methyltransferase activity towards histone H3 on lysine 4 (H3K4) and form the catalytically active hub of highly conserved protein complexes termed COMPASS. Recent genome- wide studies have revealed that MLL3 and MLL4 are frequently mutated in various forms of cancer. Similarly, Trr and Utx, a complex-specific component of the Drosophila Trr COMPASS-like complex, constitute suppressors of tissue growth. Trr acts as a major H3K4 monomethyltransferase in vivo and both Trr and MLL3 play a role in enhancer-mediated processes. Accumulating evidence also suggests that changes in enhancer activity might be one of the driving forces of carcinogenesis. However, despite the importance to maintain Trr/MLL3/MLL4 integrity for proper tissue homeostasis, very little is known about the Trr/MLL3/MLL4- dependent basic mechanisms that mediate tumor suppression. This proposal is designed to provide a training platform for the candidate to enhance his skills in working with human cells (Aim 1-2), designing, and executing high-throughput RNAi screens in Drosophila and human cells (Aim 2), to become proficient in flow cytometry and biochemical techniques (both Aim 2) and to acquire bioinformatics skills to analyze genome-wide high-throughput sequencing data (Aim 1). Based on the role of Trr/MLL3/MLL4 in enhancer-associated H3K4 monomethylation, the candidate hypothesizes that changes in the enhancer landscape of MLL3 and/or MLL4 mutant cancers constitute a driving force of tumorigenesis. All research aims of this proposal are designed to gain mechanistic insights into the role of Trr/MLL3/MLL4 in enhancer-mediated processes in general, and particularly how changes in enhancer activity might contribute to tumorigenesis. Experiments will include the identification of MLL3/MLL4-dependent enhancers with tumorigenic potential in human colon cancer cells (Aim 1). Additionally, we want to identify factors that mediate Trr/MLL3/MLL4-dependent enhancer-activity using genome-wide RNAi screens with select enhancer elements in Drosophila and human cell lines, and by using biochemical enhancer pull down approaches (Aim 2). Finally, we want to define the role of Trr/MLL3/MLL4 and its H3K4 methyltransferase activity in enhancer-mediated tumor suppression in vivo (Aim 3). Rescue experiments of trr mutants with wild-type, catalytically inactive or hyperactive trr constructs, together with the genome-wide studies, will help us to assess whether the H3K4 methyltransferase activity of Trr is vital for its role in enhancer-mediated tumor suppression. In summary, these aims will enable us to identify select factors and enhancer-mediated processes that are crucial in cancers bearing mutations in MLL3/MLL4. This will permit the development of specific pharmaceuticals that can be used for the treatment of enhancer-mediated tumorigenesis.